Ras protein is 21 kDa GTP-binding protein containing GTPase activity, and involved in cell growth and differention. Cycling of active Ras, GTP-bound forms is accomplished by the proteins' intrinsic GTPase activity and a number of accessory preteins (Bourin, H. R., Sanders, D. A., McCormick, F., Nature, 349, 117, 1991). The mammalian ras gene family contains three homologous members, H-ras, K-ras and N-ras. K-ras gene produces two different proteins K-Ras 4A and K-Ras 4B being splice variants of the same gene. Each ras-gene encodes a 21-kDa protein of either 188 (K-Ras 4B) or 189 (H-Ras, K-Ras 4A and N-Ras) amino acids residues. All three ras oncogene differ from the wild type by a single amino acid change at residue 12, 13, or 61 due to point mutation in the ras proto-oncogenes. Three mutations inhibit inherent GTPase activity of Ras protein, consequently maintained active state of Ras, GTP-binding form which produce abnormal growth signal.
It has been reported that the abnormalities of the signal transduction induces carcinogenesis. Practically oncogenic ras gene associated with these mutations has been known to be related to 30-40% of human cancers such as pancreas cancer, bladder cancer, lung cancer, skin cancer and the like. Nowadays, new attempts have been proceeded to develop anticancer agents targeted oncogenic-ras gene to inhibit carcinogenesis (Bos, J. L., Cancer Res., 49, 4682, 1989).
As described above, mammalian cells have 4 types of Ras proteins, which have very similar structure. In particular, the amino acid sequences of Ras protein are the same completely at the N-terminus from amino acid residue 1 to 86 and almost the same in ratio of 90% from amino acid residues 87 to 164. However, the amino acid sequences are very different at the C-terminus from amino acid residues 165 to end. Especially, K-Ras proteins have specific amino acid residues, lysine rich domain at the C-terminus, which discriminates K-Ras protein from N-Ras or H-Ras proteins.
Ras protein should be attached onto plasma membrane in order to show biological activities. Therefore, Ras protein should be processed for post traslational modifications by using various enzymes before it attaches onto plasma membrane. The first, the modification is catalysed by the enzyme farnesyltransferase. The enzyme covalently links a farnesyl group (a 15-carbon isoprenoid) to a cystein residue located in the carboxyl terminal CA.sub.1 A.sub.2 X motif of Ras (in which C is cystein, A.sub.1 and A.sub.2 are aliphatic amino acid, and X is methionine or serine). This is followed by hydrolysis of the A.sub.1 A.sub.2 X sequence, metylation of the terminal carboxylate group and palmitoylation of the upstream several cystein residues.
Said farnesylation is induced at the cystein residue so as to form sulfide-ether bonding. Especially, in H-Ras and N-Ras protein, palmitoylation also occurs at the other cystein residues adjacent to the C-terminus. As a result of the farnesylation, Ras protein becomes hydrophobic and can attach onto plasma membrane. The farnesyl group of Ras protein has been known to bind easily with lipid bilayer of plasma membrane.
Although all steps of the modification described above are needed in order that Ras protein attaches suitably onto cell membrane, it also has been reported that the farnesylation is enough to show inherent Ras transforming activity. Therefore, farnesyl transferase inhibitor were developed as potential anticancer agents that would block farnesylation and thus inhibit the function of oncogenic Ras (Buss, J. E. et al., Chemistry & Biology, 2, 787, 1995).
Recently, a new animal model has been developed to evaluate anticancer agents useful clinically. As a result, transgenic animals which include activated oncogene such as ras or lack tumor suppressor gene, p53 have been obtained. In particular, Leder has reported to produce transgenic animal expressing v-H-Ras protein from the MMTV (mouse mammary tumor virus) promoter (Leder, P. et al., Cell, 49, 465, 1987). Gordon has produced transgenic animal expressing N-Ras protein from the MMTV promoter (Gordon, J. W., Oncogene, 5, 1491, 1990). However, transgenic animal using activated K-Ras gene has not yet been developed.
In the meantime, the inhibition effect of FTase inhibitor on the farnesylation of Ras proteins in vitro has been estimated. It has been found that the substrate specificity of the inhibitors are different according to the amino acid sequences of the C-termini in H-Ras protein, N-Ras protein and K-Ras protein respectively. Recent reports have shown that K-Ras, whose C-terminus CVIM predicts farnesylation, can also be geranylgeranylated in vitro, and that its prenylation in cells is inhibited by a GGTaseI inhibitor. Since the mutation in K-Ras 4B are by far the most frequent in human tumors, transgenic mouse expressing K-ras oncogene actively needs to be used for evaluation in vivo efficacy of FTase inhibitors.
In order to obtain transgenic mice with mammary tumors which can be utilized in estimating inhibition effects on farnesyl transferase activity in vitro, the present invention has constructed the expression vector containing H/K-ras 4B chimeric gene and producing the chimeric fused protein at the mouse mammary gland. Then the expression vector has been microinjected into mouse one-cell embryos and the eggs have been transfered into recipient mice to produce transgenic mice. And in order to identify the incorporation of H/K-ras 4B gene into genomic DNA sequence, polymerase chain reaction (PCR) and Southern blot were accomplished finally. Additionally, the expression of H/K-ras 4B genes was confirmed by RT-PCR and Northern blot in specific tissues.